Project Summary/Abstract The ultimate goal of this phase II STTR proposal is to expand the capabilities of SAB's diversitAb? platform by continuing advanced development of Transchromosomic goats (TcGs). This will be accomplished by producing male and female goat endogenous immunoglobulin gene knockout cell lines that contain SAB Capra's human artificial chromosome (HAC) which encodes the entire repertoire of the germline human antibody genes thereby greatly improving the production efficiency of fully human antibodies in TcGs. To further advance the TcG platform towards commercial production of therapeutic and diagnostic antibody products, optimization of the antibody purification process, development of quality control assays, and further development of a purified TcG- derived pandemic influenza antibody product targeting H7N9 by completing in-vitro and in-vivo evaluation is also proposed. To accomplish these goals, endogenous immunoglobulin gene knockouts will be facilitated utilizing the CRISPR/Cas9 system in domestic Nubian/Boar goat fetal fibroblast (GFF) cells, and transfer of the SAB- designed HAC into the knockout cells will be accomplished by microcell mediated chromosome transfer (MMCT). HAC-containing knockout GFF cells will be used to produce embryos by somatic cell nuclear transfer (SCNT). After gestation and birth of multiple TcGs, molecular characterization will be completed, presence of the knockouts and HAC will be confirmed, and human IgG production will be confirmed and evaluated. Two of these TcGs will then be hyperimmunized with a plasmid DNA vaccine targeting H7N9. Plasma will be collected from the hyperimmunized TcGs, and anti-H7N9 fully human IgG will be purified from the TcG plasma. The in-vitro potency and in-vivo efficacy of the purified TcG-derived anti-H7N9 human IgG product will then be evaluated. Optimization of established purification procedures will be undertaken to ensure efficient and effective purification of TcG-derived human IgG from plasma, and development of quality control assays to evaluate product identity, purity, and potency will be completed. SAB's diversitAb? platform for human antibody production is currently utilized in transchromosomic bovines against a wide range of antigens including viruses, bacteria, oncology targets, recombinant proteins, and DNA vaccines. This proven technology has the advantages of scalability, simplicity, and broad applicability. The addition of TcGs to the platform allows for simpler and cheaper rapid-response production of small volume targeted products as well as diagnostic reagents for serological testing of emerging infectious diseases.